Pharmacological characterization of cloned human NK-2 (neurokinin A) receptor expressed in a baculovirus/Sf-21 insect cell system
Aharony, D.; Little, J.; Powell, S.; Hopkins, B.; Bundell, K.R.; McPheat, W.L.; Gordon, R.D.; Hassall, G.; Hockney, R.; Griffin, R.
Molecular Pharmacology 44(2): 356-363
1993
ISSN/ISBN: 0026-895X PMID: 8394992 Document Number: 415326
Using the novel ligand (4,5-3H-Leu-9)neurokinin A ((4,5-3H-Leu-9) NKA) in a receptor binding assay, we characterized the pharmacology of a cloned neurokinin NK-2 receptor from human lung (hNK-2R), expressed in baculovirus-infected Sf-21 insect cells. Functional hNK-2R cDNA clones were isolated from human lung using a polymerase chain reaction-based methodology. hNK-2R was cloned into pAcYM1, a vector designed to couple expression to the polyhedrin promoter, and the recombinant baculovirus was isolated and used to infect Sf-21 insect cells. hNK-2R expression levels were monitored by Northern blots and 125-I-NKA binding assays. Isolates demonstrating the highest specific binding of 125-I-NKA were grown and membrane preparations from high-speed centrifugations were prepared from both hNK-2R-expressing and wild-type virus-infected cells. (3H)NKA bound in a protein-dependent, saturable (B-max = 820 +- 167 fmol/mg of protein), and highly specific (88 +- 5%) manner to hNK-2R, but not to membranes from cells infected with wild-type virus (14 +- 8%, 7 +- 1 0 fmol/mg of protein). (3H)NKA binding was rapid (k-1 = 0.085 nM-1 cntdot min-1) and reversible (t-1/2 = 4-5 min). Equilibrium binding experiments demonstrated binding to a mixture of receptors in high and low affinity states (K-d1 = 2.28 +- 0.26 nM and K-d2 = 266 +- 91 nM). Binding to hNK-2R was greatly enhanced (400%-600%) by Ca-2+ and Mg-2+ (EC-50 values of 30 mu-M and 140 mu-M, respectively), whereas guanosine-5'-O-(3'-thio)triphosphate and guanosine-5'-(beta,gamma-imido)diphosphate were inhibitory. Competition experiments with agonists also demonstrated binding to high and low affinity states, with the following order of potency: NKA gt (Nle-10)NKA(4-10) gt (beta-Ala-8)NKA(4-10) mchgt substance P; Senktide and the NK-1 antagonist CP96,345 (10 mu-m) did not inhibit binding. Inhibition of binding by selective NK-2 antagonists was consistent with a single affinity state and demonstrated the following order of affinity: SR48,968 mchgt MEN10,376 gt L659,877 gt R396. These data suggest that infection of Sf-21 cells with baculovirus expression vector harboring the cDNA of hNK-2R resulted in expression of high affinity, G protein-coupled hNK-2R, with pharmacological selectivity compatible with the NK-2A receptor subtype.